Winter cherry tissue culture method

Abstract

The invention provides a winter cherry tissue culture method, and belongs to the technical field of plant tissue culture. According to the method, a winter cherry semi-lignified stem section is used as an explant, and a winter cherry tissue culture and rapid propagation system is established. According to the method, the formula of an initial culture medium of a winter cherry stem segment is MS + 1.0 mg / L 6-BA + 0.1 mg / L IBA; the most suitable proliferation culture medium formula is MS + 0.3 mg / L 6-BA + 0.2 mg / L IBA, and the proliferation coefficient is 4.6; the optimal rooting culture medium formula is 1 / 2MS + 0.5 mg / LNAA, under the optimal culture condition, the rooting rate of winter cherry tissue culture is 84%, the average root number is 6.24, and the transplanting survival rate is 92%. Reference is provided for tissue culture and rapid propagation of the winter cherry, and a foundation is laid for genetic engineering research of the winter cherry.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a winter cherry blossom tissue culture method. Background Art

[0002] Winter cherry (Cerasus cerasoides (D.Don) Sok.) is also known as winter crabapple and Indian spring, named because it blooms in winter. Winter cherry belongs to the genus Cerasus in the Rosaceae family. It is a deciduous tree and the only species of cherry blossom in my country's wild cherry resources that blooms in winter. Winter cherry is a wild native tree species unique to Yunnan. It has strong resistance to adversity and strong adaptability. It is mainly distributed in hillsides, valleys, streams and jungles in high-altitude areas. Winter cherry is drought-resistant, barren-resistant, has strong affinity and a high survival rate. In addition to being an ornamental tree species, it can also be used as a rootstock for cherry plants. Therefore, winter cherry is a very good cherry germplasm resource.

[0003] At present, the research on winter cherry blossoms mainly focuses on the characteristics of winter cherry blossom community, eco-geography, flowering habits, phenology, viewing period and reproduction. Winter cherry blossoms are mainly propagated by seeds, and can also be propagated by asexual methods such as cuttings and grafting. However, due to the quality of the seeds themselves, sowing conditions and management conditions, seed propagation has problems such as low germination rate and uneven seedling emergence. Cutting propagation is restricted by seasons, requires a large number of branches, and has problems such as high difficulty in cutting technology. Therefore, cutting propagation cannot obtain a large number of seedlings in a short period of time and cannot meet market needs. Compared with traditional propagation methods, tissue culture rapid propagation technology is not affected by seasons, can shorten the breeding cycle, improve the reproduction coefficient, meet the needs of production and promotion, and ensure the stable inheritance of winter cherry blossoms. Although the application value of winter cherry blossoms is extremely high, there are few related studies on tissue culture rapid propagation of winter cherry blossoms. Existing reports use the pedicels, receptacles, ovaries, leaves and buds of winter cherry blossoms as explants to study the effects of hormone concentration on the healing rate of explants and measures to inhibit browning. However, there is no report on establishing a stable tissue culture and rapid propagation system of Prunus hirta using semi-lignified stem segments of Prunus hirta as explants. Summary of the invention

[0004] In view of this, the present invention provides a winter cherry tissue culture method, takes the semi-lignified stem segments of winter cherry as explants, establishes a stable tissue culture and rapid propagation system of winter cherry, and provides a reference for the tissue culture and rapid propagation of winter cherry.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for tissue culture of winter cherry, comprising the following steps:

[0007] S1. Explant preparation: Cut the semi-lignified shoots of winter cherry, remove the leaves, cut into single-bud stem segments, and then disinfect them;

[0008] S2. Primary culture: inoculate the sterilized stem segments with buds into the primary culture medium to induce axillary bud germination; the formula of the primary culture medium is: MS medium + 0.8-1.2 mg / L 6-BA + 0.08-0.12 mg / L IBA + 18-22 g / L sucrose + 6-7 g / L agar;

[0009] S3. Subculture: When the axillary buds grow to more than 1 cm, cut the stem tips of 0.8-1.0 cm in length and inoculate them into subculture medium for subculture; the formula of subculture medium is: MS medium + 0.25-0.35 mg / L 6-BA + 0.18-0.22 mg / L IBA;

[0010] S4. Rooting culture: After 24 to 26 days of subculture, the subculture seedlings are transferred to a rooting medium for rooting culture; the formula of the rooting medium is: 1 / 2MS medium + 0.4 to 0.6 mg / L NAA;

[0011] S5. After 24 to 26 days of rooting culture, the winter cherry tissue culture seedlings with adventitious root length ≥ 2 cm are transplanted and hardened.

[0012] Preferably, the disinfection treatment is: firstly, stirring and soaking with detergent, then soaking with alcohol solution, and then soaking with sodium hypochlorite solution.

[0013] Preferably, the detergent is stirred and soaked for 17 to 23 minutes, and then rinsed with water for 40 to 60 minutes.

[0014] Preferably, the volume fraction of the alcohol solution is 70-80%, and the immersion time of the alcohol solution is 25-35 seconds.

[0015] Preferably, the mass fraction of the sodium hypochlorite solution is 4-6%, and the soaking time of the sodium hypochlorite solution is 9-11 minutes.

[0016] Preferably, during transplanting, the root culture medium is washed off with water, the roots are dipped in a carbendazim solution, and the seeds are transplanted into a seedling medium for cultivation.

[0017] Preferably, the root dipping treatment lasts for 2 to 3 minutes.

[0018] Preferably, the seedling growing matrix includes perlite, peat soil and vermiculite.

[0019] Preferably, the volume ratio of perlite, peat soil and vermiculite is 1:3:2.

[0020] Preferably, the temperatures for primary culture, subculture and rooting culture are 24-26° C., and the light / dark cycle is 16 h / 8 h.

[0021] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention uses the semi-lignified stem segments of winter cherry as explants to establish a winter cherry tissue culture rapid propagation system. In the present invention, the initial culture medium formula of winter cherry stem segments is MS+1.0mg / L 6-BA+0.1mg / L IBA; the optimal proliferation culture medium formula is MS+0.3mg / L 6-BA+0.2mg / LIBA, and the proliferation coefficient is 4.6; the optimal rooting culture medium formula is 1 / 2MS+0.5mg / LNAA. Under the optimal culture conditions, the rooting rate of winter cherry tissue culture is 84%, the average number of roots is 6.24, and the transplant survival rate is 92%. The present invention provides a reference for the tissue culture rapid propagation of winter cherry, and also lays a foundation for the genetic engineering research of winter cherry. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram of the process of establishing seedlings from semi-lignified stem segments of winter cherry, where 1-A is a winter cherry seedling, 1-B is a semi-lignified stem segment of winter cherry inoculated into the primary culture medium, 1-C is the germination of axillary buds of the explant, and 1-D is the elongation of axillary buds of the explant to form green shoots.

[0023] Figure 2 The growth conditions of winter cherry blossoms on different basic culture media, among which 2-A uses MS as the basic culture medium, 2-B uses QL as the basic culture medium, and 2-C uses WPM as the basic culture medium.

[0024] Figure 3 The effects of different growth regulators on rooting. DETAILED DESCRIPTION

[0025] The present invention provides a method for tissue culture of winter cherry, comprising the following steps:

[0026] S1. Explant preparation: Cut the semi-lignified shoots of winter cherry, remove the leaves, cut into single-bud stem segments, and then disinfect them;

[0027] S2. Primary culture: inoculate the sterilized stem segments with buds into the primary culture medium to induce axillary bud germination; the formula of the primary culture medium is: MS medium + 0.8-1.2 mg / L 6-BA + 0.08-0.12 mg / L IBA + 18-22 g / L sucrose + 6-7 g / L agar;

[0028] S3. Subculture: When the axillary buds grow to more than 1 cm, cut the stem tips of 0.8-1.0 cm in length and inoculate them into subculture medium for subculture; the formula of subculture medium is: MS medium + 0.25-0.35 mg / L 6-BA + 0.18-0.22 mg / L IBA;

[0029] S4. Rooting culture: After 24 to 26 days of subculture, the subculture seedlings are transferred to a rooting medium for rooting culture; the formula of the rooting medium is: 1 / 2MS medium + 0.4 to 0.6 mg / L NAA;

[0030] S5. After 24 to 26 days of rooting culture, the winter cherry tissue culture seedlings with adventitious root length ≥ 2 cm are transplanted and hardened.

[0031] The present invention first cuts the semi-lignified new shoots of winter cherry, removes the leaves, cuts them into single bud stem segments, and obtains explants. The explants are preferably cut in spring. Then the explants are disinfected. First, they are stirred and soaked with detergent. The stirring and soaking time is 17 to 23 minutes, more preferably 19 to 21 minutes, and more preferably 20 minutes. After the soaking is completed, they are rinsed with water for 40 to 60 minutes, more preferably 45 to 55 minutes, and more preferably 50 minutes; preferably, tap water is used for rinsing. After the rinsing is completed, the explants are transferred to a clean bench and soaked with an alcohol solution. The volume fraction of the alcohol solution is 70 to 80%, more preferably 72 to 78%, and more preferably 75%; the alcohol soaking time is 25 to 35 seconds, more preferably 28 to 32 seconds, and more preferably 30 seconds. After soaking in the alcohol solution, rinse with sterile water, and then soak in a sodium hypochlorite solution, wherein the mass fraction of the sodium hypochlorite solution is 4-6%, and more preferably 5%; the soaking time of the sodium hypochlorite solution is 9-11 minutes, more preferably 9.5-10.5 minutes, and more preferably 10 minutes, and then rinse with sterile water for 5-6 times, and absorb the moisture on the surface of the stem segment, preferably using sterile filter paper to absorb the moisture.

[0032] The present invention inoculates the disinfected bud stem segment into the primary culture medium to induce axillary bud germination. The formula of the primary culture medium is: MS culture medium + 0.8-1.2 mg / L 6-BA + 0.08-0.12 mg / L IBA + 18-22 g / L sucrose + 6-7 g / L agar; in the primary culture medium, the concentration of 6-BA is more preferably 0.9-1.1 mg / L, more preferably 1 mg / L; the concentration of IBA is more preferably 0.09-0.11 mg / L, more preferably 0.1 mg / L; the concentration of sucrose is more preferably 19-21 g / L, more preferably 20 g / L; the content of agar is more preferably 6.2-6.8 g / L, more preferably 6.5 g / L.

[0033] In the present invention, when the induced axillary buds grow to more than 1.0 cm, 0.8-1.0 cm long shoot tips are cut and inoculated into a subculture medium for subculture. The formula of the subculture medium is: MS medium + 0.25-0.35 mg / L 6-BA + 0.18-0.22 mg / L IBA; in the subculture medium, the concentration of 6-BA is further preferably 0.28-0.32 mg / L, more preferably 0.3 mg / L; the concentration of IBA is further preferably 0.19-0.21 mg / L, more preferably 0.2 mg / L.

[0034] In the present invention, after 24 to 26 days of subculture, the subculture seedlings are transferred to a rooting medium for rooting culture; the formula of the rooting medium is: 1 / 2MS medium + 0.4 to 0.6 mg / L NAA; the concentration of the NAA is further preferably 0.45 to 0.55 mg / L, and more preferably 0.5 mg / L.

[0035] After 24 to 26 days of rooting culture, the winter cherry tissue culture seedlings with adventitious root length ≥ 2 cm are transplanted and hardened. During transplanting, the tissue culture seedlings are taken out from the culture medium, preferably with tweezers, the root culture medium is washed off with water, the roots are dipped in a carbendazim solution, and the seedlings are transplanted into the seedling medium and covered with a film for culture; the dilution multiple of the carbendazim is 400 to 600 times, more preferably 450 to 550 times, and more preferably 500 times; the root dipping time is 2 to 3 minutes, the seedling medium includes perlite, peat soil and vermiculite, the volume ratio of perlite, peat soil and vermiculite is 1:3:2, and the purpose of the film is to keep warm and moist.

[0036] The temperatures for primary culture, subculture and rooting culture are 24-26°C, more preferably 25°C; the light / dark cycle is 16h / 8h.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] 1. Explant acquisition and disinfection

[0040] In spring, the semi-lignified shoots of winter cherry were cut, the leaves were removed, and the shoots were cut into single-bud stem segments to obtain explants. The explants were disinfected. First, they were stirred and soaked in detergent for 20 minutes, then rinsed with tap water for 50 minutes, transferred to a clean bench, soaked in a 75% alcohol solution for 30 seconds, rinsed with sterile water three times, and then soaked in a 5% sodium chlorate solution for 10 minutes, then washed with sterile water five times, and the surface moisture of the stem segments was dried with sterile filter paper.

[0041] 2. Primary Culture

[0042] The sterilized bud stem segments were inoculated into the primary culture medium to induce axillary bud germination. The formula of the primary culture medium is: MS medium + 1.0 mg / L 6-BA + 0.1 mg / L IBA + 18-22 g / L sucrose + 6.5 g / L agar, pH 5.8. The culture temperature is (25±1)℃, and the light / dark cycle is 16h / 8h.

[0043] 3. Subculture

[0044] When the induced axillary buds grow to more than 1.0 cm, cut the stem tips with the same growth and inoculate them into the subculture medium. The length of the cut is 1.0 cm. The formula of the subculture medium is: MS medium + 0.3 mg / L 6-BA + 0.2 mg / L IBA. The culture conditions are the same as the primary culture.

[0045] 4. Rooting Induction Culture

[0046] After 25 days of subculture, subculture seedlings with consistent growth were selected and transferred to rooting medium for rooting induction culture. The formula of rooting medium was: 1 / 2MS medium + 0.5 mg / L NAA. The culture conditions were the same as those of primary culture.

[0047] 5. Hardening and transplanting

[0048] The strong winter cherry blossom tissue culture seedlings with adventitious root length ≥ 2cm were acclimatized. When transplanting, the tissue culture seedlings were gently taken out with tweezers, the root culture medium was washed off with clean water, and then the roots were treated with 500 times carbendazim solution for 3 minutes, and then transplanted into the sterilized seedling medium, covered with film to keep moisture. The seedling medium is composed of perlite, peat soil and vermiculite, and the volume ratio of perlite, peat soil and vermiculite is 1:3:2. The survival rate was counted 30 days after transplanting, and the survival rate of winter cherry blossom transplanted seedlings was 92%. The transplanted seedlings grew robustly, with green leaves and good growth conditions.

[0049] Comparative Example 1

[0050] The difference from Example 1 is that the disinfection time of 5% sodium hypochlorite is 5 minutes, and the other culture conditions are the same as those in Example 1.

[0051] Comparative Example 2

[0052] The difference from Example 1 is that the disinfection time of 5% sodium hypochlorite is 8 minutes, and the other culture conditions are the same as those in Example 1.

[0053] Comparative Example 3

[0054] The difference from Example 1 is that the disinfection time of 5% sodium hypochlorite is 15 minutes, and the other culture conditions are the same as those in Example 1.

[0055] Comparative Example 4

[0056] The difference from Example 1 is that the subculture medium uses QL as the basal medium, and the other culture conditions are the same as those in Example 1.

[0057] Comparative Example 5

[0058] The difference from Example 1 is that the subculture medium uses WPM as the basic medium, and other culture conditions are the same as those in Example 1.

[0059] Comparative Example 6

[0060] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.2 mg / L, the concentration of IBA is 0.05 mg / L, and the other culture conditions are the same as those in Example 1.

[0061] Comparative Example 7

[0062] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.2 mg / L, the concentration of IBA is 0.1 mg / L, and the other culture conditions are the same as those in Example 1.

[0063] Comparative Example 8

[0064] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.2 mg / L, the concentration of IBA is 0.2 mg / L, and the other culture conditions are the same as those in Example 1.

[0065] Comparative Example 9

[0066] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.3 mg / L, the concentration of IBA is 0.05 mg / L, and the other culture conditions are the same as those in Example 1.

[0067] Comparative Example 10

[0068] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.3 mg / L, the concentration of IBA is 0.1 mg / L, and the other culture conditions are the same as those in Example 1.

[0069] Comparative Example 11

[0070] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.5 mg / L, the concentration of IBA is 0.05 mg / L, and the other culture conditions are the same as those in Example 1.

[0071] Comparative Example 12

[0072] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.5 mg / L, the concentration of IBA is 0.1 mg / L, and the other culture conditions are the same as those in Example 1.

[0073] Comparative Example 13

[0074] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.5 mg / L, the concentration of IBA is 0.2 mg / L, and the other culture conditions are the same as those in Example 1.

[0075] Comparative Example 14

[0076] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.8 mg / L, the concentration of IBA is 0.05 mg / L, and the other culture conditions are the same as those in Example 1.

[0077] Comparative Example 15

[0078] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.8 mg / L, the concentration of IBA is 0.1 mg / L, and the other culture conditions are the same as those in Example 1.

[0079] Comparative Example 16

[0080] The difference from Example 1 is that the concentration of 6-BA in the proliferation medium is 0.8 mg / L, the concentration of IBA is 0.2 mg / L, and the other culture conditions are the same as those in Example 1.

[0081] Comparative Example 17

[0082] The difference from Example 1 is that NAA is replaced by IBA in the rooting medium, and the concentration of IBA is 0.5 mg / L.

[0083] Comparative Example 18

[0084] The difference from Example 1 is that NAA is replaced by IBA in the rooting medium, and the concentration of IBA is 1.0 mg / L.

[0085] Comparative Example 19

[0086] Different from Example 1, the concentration of NAA in the rooting medium is 1.0 mg / L.

[0087] Experimental Example 1. Effect of disinfection time on contamination and survival of winter cherry stem segments

[0088] Sowing winter cherry seeds in the greenhouse to obtain seedlings ( Figure 11-A), take the semi-lignified stem segments of winter cherry, sterilize the explants according to the methods of Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3, and then inoculate them into the primary culture medium. Observe the growth of the explants, and calculate the contamination rate and survival rate after 28 days. Contamination rate (%) = (number of contaminated explants / number of inoculated explants) × 100%; survival rate (%) = (number of surviving explants / number of inoculated explants) × 100%. The results are as follows Figure 1 And as shown in Table 1.

[0089] The growth of the explants in Example 1 is as follows Figure 1 As shown, Figure 2 1-B in the figure is to inoculate the explants in the primary culture medium, and axillary buds will germinate after 2 weeks ( Figure 1 1-C), after 4 weeks, the axillary buds elongate to form green shoots ( Figure 1 The results showed that MS medium supplemented with 1.0 mg / L 6-BA and 0.1 mg / L IBA was suitable for the initiation and differentiation of axillary buds of winter cherry stem segments, and there was no vitrification phenomenon in the new buds.

[0090] Table 1 Effects of disinfection time on contamination and survival of winter cherry stem segments

[0091]

[0092] Note: Different letters in the same column indicate significant differences at the α=0.05 level.

[0093] As shown in Table 1, different disinfection times have a significant effect on the contamination and survival rate of winter cherry stem segments. As the disinfection time of sodium hypochlorite increases, the contamination rate decreases, but the survival rate shows a trend of first increasing and then decreasing. Therefore, the best disinfection time for winter cherry is to disinfect with 75% alcohol for 30 seconds and then with 5% sodium hypochlorite for 10 minutes.

[0094] Experimental Example 2. Effect of basic culture medium on subculture

[0095] Explants were sterilized and primary cultured according to the method of Example 1 to obtain sterile tissue culture seedlings. When the axillary buds grew to more than 1 cm, 1.0 cm stem tips were cut and inoculated into secondary culture medium with MS, QL or WPM as the basic culture medium. 0.5 mg / L 6-BA was added to the three basic culture mediums; 15 plants were treated each time and repeated 3 times. After 28 days, the growth of the plants was observed and counted. The results are as follows: Figure 2 The results showed that the proliferation coefficient was the highest when MS was used as the basic culture medium, and the tissue culture seedlings proliferated in clusters, but some plants were weak ( Figure 2 2-A in the above). With QL and WPM as the basic culture medium, the proliferation coefficient is low. The leaves of the tissue culture seedlings grown on the QL medium are green and the growth is good ( Figure 22-B in the medium), while the plantlets grown on WPM medium grew slowly and were short ( Figure 2 Therefore, MS medium with high proliferation coefficient was selected as the basic medium for subsequent experiments.

[0096] Experimental Example 3. Effects of different growth regulators on proliferation culture

[0097] Explant disinfection and primary culture were performed according to the method of Example 1 to obtain sterile tissue culture seedlings. When the axillary buds grew to more than 1 cm, 1.0 cm stem tips were cut and inoculated into different proliferation culture media shown in Table 2, 20 plants were treated in each treatment, and repeated 3 times. After 28 days, the proliferation of tissue culture seedlings was investigated after 28 days of culture, and their growth conditions were recorded. The results are shown in Table 2. Proliferation coefficient = total number of proliferated plants / number of inoculated plants.

[0098] Table 2 Effects of different growth regulators on proliferation

[0099]

[0100]

[0101] Note: Different letters in the same column indicate significant differences at the α=0.05 level.

[0102] Experimental Example 4. Effects of different growth regulators on rooting

[0103] According to the method of Example 1, explant disinfection, primary culture and subculture were carried out. After 25 days of subculture, subculture seedlings with consistent growth were selected and transplanted into different rooting mediums shown in Table 3. Five bottles were treated for each treatment, and 6 plants were inoculated in each bottle. Each treatment was repeated 3 times. After 25 days, the rooting situation was statistically analyzed, including rooting rate, average number of roots and average root length. Rooting rate (%) = (number of rooted plants / number of inoculated plants) × 100%, average number of roots = total number of roots / total number of rooted plants, average root length = total root length / total number of roots of plants, and the results are shown in Table 3.

[0104] Table 3 Effects of different growth regulators on rooting

[0105]

[0106] Note: Different letters in the same column indicate significant differences at the α=0.05 level.

[0107] The results showed that winter cherry blossoms could take root in 1 / 2MS rooting medium containing IBA or NAA, but when NAA was added, the rooting rate and number of roots of winter cherry blossoms were better than those in the medium containing IBA. When the NAA concentration was 0.5 mg / L, the rooting rate was as high as 84%, and the roots were thick. When the NAA concentration increased to 1.0 mg / L, the rooting rate decreased, the roots were shorter, and callus tissue appeared on the roots ( Figure 3 ). When IBA was added, the taproot was longer and could produce lateral roots, but the rooting rate and number of roots were not high. There was little difference in the rooting rate and number of roots when the IBA concentration was 0.5mg / L and 1.0mg / L. Comprehensive analysis of the rooting rate, number of roots and root growth showed that the optimal rooting medium for winter cherry blossoms was 1 / 2MS with 0.5mg / L NAA.

[0108] It can be seen from the above embodiments that the present invention provides a method for tissue culture of winter cherry, which establishes a stable tissue culture and rapid propagation system of winter cherry and provides a reference for tissue culture and rapid propagation of winter cherry.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for tissue culture of winter cherry, characterized in that: The following steps are involved: S1. Explant preparation: Cut the semi-lignified shoots of winter cherry, remove the leaves, cut into single-bud stem segments, and then disinfect them; S2. Primary culture: inoculate the sterilized stem segments with buds into the primary culture medium to induce axillary bud germination; the formula of the primary culture medium is: MS medium + 0.8-1.2 mg / L 6-BA + 0.08-0.12 mg / L IBA + 18-22 g / L sucrose + 6-7 g / L agar; S3. Subculture: When the axillary buds grow to more than 1 cm, cut the stem tips of 0.8 to 1.0 cm in length and inoculate them into subculture medium for subculture; the formula of the subculture medium is: MS medium + 0.25 to 0.35 mg / L 6-BA + 0.18 to 0.22 mg / L IBA; S4. Rooting culture: After 24 to 26 days of subculture, the subculture seedlings are transferred to a rooting medium for rooting culture; the formula of the rooting medium is: 1 / 2MS medium + 0.4 to 0.6 mg / L NAA; S5. After 24 to 26 days of rooting culture, the winter cherry tissue culture seedlings with adventitious root length ≥ 2 cm are transplanted and hardened.

2. The winter cherry tissue culture method according to claim 1, characterized in that: The disinfection treatment is as follows: firstly, the mixture is stirred and soaked with detergent, then soaked with alcohol solution, and then soaked with sodium hypochlorite solution.

3. The winter cherry tissue culture method according to claim 2, characterized in that: The detergent is stirred and soaked for 17 to 23 minutes, and then rinsed with water for 40 to 60 minutes.

4. The winter cherry tissue culture method according to claim 2, characterized in that: The volume fraction of the alcohol solution is 70-80%, and the soaking time of the alcohol solution is 25-35 seconds.

5. The winter cherry tissue culture method according to claim 2, characterized in that: The mass fraction of the sodium hypochlorite solution is 4-6%, and the soaking time of the sodium hypochlorite solution is 9-11 minutes.

6. The method for tissue culture of winter cherry blossoms according to claim 5, characterized in that: When transplanting, wash off the root culture medium with water, dip the roots in carbendazim solution, and transplant them into the seedling medium for cultivation.

7. The winter cherry tissue culture method according to claim 6, characterized in that: The root dipping treatment lasts for 2 to 3 minutes.

8. The method for tissue culture of winter cherry blossoms according to claim 6 or 7, characterized in that: The seedling raising matrix comprises perlite, peat soil and vermiculite.

9. The winter cherry tissue culture method according to claim 8, characterized in that: The volume ratio of the perlite, peat soil and vermiculite is 1:3:

2.

10. The winter cherry tissue culture method according to claim 1, characterized in that: The temperatures for primary culture, subculture and rooting culture were 24-26°C, and the light / dark cycle was 16h / 8h.

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